Until about a decade ago, resolution in far-field light microscopy was thought to be limited to ˜200-250 nanometers in the focal plane, concealing details of sub-cellular structures and constraining its biological applications. Breaking this diffraction barrier by the seminal concept of stimulated emission depletion (“STED”) microscopy has made it possible to image biological systems at the nanoscale with light. Additional details are provided in an article titled “Far-Field Optical Nanoscopy by Stefan W. Hell (316 Science, 1153-1158, May 25, 2007), which is incorporated herein by reference in its entirety. STED microscopy and other members of reversible saturable optical fluorescence transitions (“RESOLFT”) family achieve a resolution >10-fold beyond the diffraction barrier by engineering the microscope's point-spread function (“PSF”) through optically saturable transitions of the (fluorescent) probe molecules.
Lately, an emerging group of localization-based techniques has obtained similar resolution in the lateral plane. This group includes fluorescence photoactivation localization microscopy (“FPALM”), photoactivation localization microscopy (“PALM”), stochastic optical reconstruction microscopy (“STORM”), and PALM with independently running acquisition (“PALMIRA”). FPALM is described in more detail in an article titled “Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy” by Samuel T. Hess et al. (91 Biophysical Journal, 4258-4272, December 2006), which is incorporated herein by reference in its entirety. PALM is described in more detail in an article titled “Imaging Intracellular Fluorescent Proteins at Nanometer Resolution” by Eric Betzig et al. (313 Science, 1642-1645, Sep. 15, 2006), which is incorporated herein by reference in its entirety. STORM is described in more detail in an article titled “Sub-Diffraction-Limit Imaging by Stochastic Optical Reconstruction Microscopy” by Michael J. Rust et al. (Nature Methods/Advance Online Publication, Aug. 9, 2006), which is incorporated herein by reference in its entirety. PALMIRA is described in more detail in an article titled “Resolution of λ/10 in Fluorescence Microscopy Using Fast Single Molecule Photo-Switching” by H. Bock et al. (88 Applied Physics A, 223-226, Jun. 1, 2007), and an article titled “Photochromic Rhodamines Provide Nanoscopy With Optical Sectioning” by J. Folling et al. (Angew. Chem. Int. Ed., 46, 6266-6270, 2007), each of which is incorporated herein by reference in its entirety. As referred to in the current application, the term photo-sensitive refers to both photo-activatable (e.g., switching probes between an on state and an off state) and photo-switching (e.g., switching between a first color and a second color).
While utilizing similar optical switching mechanisms, this latter group of microscopes circumvents the diffraction limit by basing resolution improvement on the precise localization of spatially well-separated fluorescent molecules, a method previously used to track, for example, conventionally labeled myosin V molecules with 1.5 nanometers localization accuracy. This method is described in more detail in an article titled “Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging With 1.5-nanometers Localization” by Ahmet Yildiz et al. (300 Science, 2061-2065, Jun. 27, 2003), which is incorporated herein by reference in its entirety.
To resolve complex nanoscale structures by localization-based methods, the sample is labeled with photo-sensitive probes, such as photo-activatable (“PA”) fluorescent probes (e.g., PA proteins or caged organic dyes). Activation of only a sparse subset of molecules at a time allows their separate localization. By repeated bleaching or deactivation of the active molecules in concert with activation of other inactive probe molecules, a large fraction of the whole probe ensemble can be localized over time. The final sub-diffraction image of the labeled structure is generated by plotting the positions of some or all localized molecules.
Based on the rapid development in both RESOLFT and localization-based techniques, the impact of super-resolution far-field fluorescence microscopy on the biological sciences is expected to increase significantly. Within 2007 alone subdiffraction multi-color imaging has been reported for the first time for STED microscopy, PALMIRA, STORM, and FPALM has successfully been demonstrated in live cells. Some of these reports are included in an article titled “Two-Color Far-Field Fluorescence Nanoscopy” by Gerald Donnert et al. (Biophysical Journal, L67-L69, Feb. 6, 2007), in an article by M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang (Science 317, 1749-1753, 2007), and in an article titled “Dynamic Clustered Distribution of Hemagglutinin Resolved at 40 nanometers in Living Cell Membranes Discriminates Between Raft Theories” by Samuel T. Hess et al. (Proc. Natl. Acad. Sci. USA 104, 17370-17375, Oct. 30, 2007), each of which is incorporated herein by reference in its entirety.
However, the slow progress in 3D super-resolution imaging has limited the application of these techniques to two-dimensional (“2D”) imaging. The best 3D resolution until recently had been 100 nanometers axially at conventional lateral resolution. Achieved by the combination of two objective lens apertures in 4Pi microscopy, it has been applied for more than a decade. This is described in more detail in an article titled “H2AX Chromatin Structures and Their Response to DNA Damage Revealed by 4Pi Microscopy” by Joerg Bewersdorf et al. (Proc. Natl. Acad. Sci. USA 103, 18137-18142, Nov. 28, 2006), which is incorporated by reference in its entirety. Only lately first 3D STED microscopy images have been published exceeding this resolution moderately with 139 nanometer lateral and 170 nanometer axial resolution. These images are presented in more detail in an article by K. I. Willig, B. Harke, R. Medda, and S. W. Hell (Nat. Methods 4, 915-918, 2007), which is incorporated by reference in its entirety. While this represents a ˜10-fold smaller resolvable volume than provided by conventional microscopy, it is still at least 10-fold larger than a large number of sub-cellular components, for example synaptic vesicles. Recently, an article (Huang et al., Science 2008) has reported first 3D STORM of thin optical sections (<600 nanometers) with sub-100 nanometer 3D resolution under reducing (low oxygen) conditions.
Moreover, current understanding of fundamental biological processes on the nanoscale (e.g., neural network formation, chromatin organization) is limited because these processes cannot be visualized at the necessary sub-millisecond time resolution. Current biological research at the sub-cellular level is constrained by the limits of spatial and temporal resolution in fluorescence microscopy. The diameter of most organelles is below the diffraction limit of light, limiting spatial resolution and concealing sub-structure. Recent developments (e.g., STED, FPALM, STORM, etc.) have dramatically enhanced the spatial resolution and even overcome the traditional diffraction barrier. However, comparable improvements in temporal resolution are still needed.
Particle-tracking techniques can localize small objects (typically<diffraction limit) in live cells with sub-diffraction accuracy and track their movement over time. But conventional particle-tracking fluorescence microscopy cannot temporally resolve interactions of organelles, molecular machines, or even single proteins, which typically happen within milliseconds.
The spatial localization accuracy of single particles in a fluorescence microscope is approximately proportional to d/√{square root over (N)} (d=spatial resolution; N=total number of detected fluorescence photons from the particle) in the absence of background and effects due to finite pixel size. For longer acquisition times more signal can be accumulated, hence increased temporal resolution requires a trade-off of decreased spatial localization accuracy. For bright organelles containing a few hundred fluorescent molecules, (or future fluorescent molecules with increased brightness), sufficient signal can be accumulated quickly. However, especially for 3D localization where data acquisition is far more complicated than in 2D, technical constraints arising from axial scanning and/or camera readout times limit the recording speed, and therefore, the temporal resolution.
For example, a particular 3D particle-tracking technique can track particles only with 32 milliseconds time resolution. This technique scans a 2-photon excitation focus in a 3D orbit around the fluorescent particle and determines its 3D position by analyzing the temporal fluorescence fluctuations. The temporal resolution is ultimately limited by the frequency with which the focus can revolve in 3D around the particle. This technique is described in more detail in an article titled “3-D Particle Tracking In A Two-Photon Microscope: Application To The Study Of Molecular Dynamics IN Cells” by V. Levi, Q. Ruan, and E. Gratton (Biophys. J., 2005, 88(4): pp. 2919-28), which is incorporated by reference in its entirety.
In another example, another current 3D particle-tracking technique combines traditional particle-tracking with widefield “bifocal detection” images. Particles are simultaneously detected in one plane close to the focal plane of the particle and a second plane 1 micrometer out of focus. The lateral and axial coordinates are derived from the 2 images. In accordance with this technique, the temporal resolution is limited to the 2-50 milliseconds range, and the localization accuracy is limited to the 2-5 nanometer range. Additional details are described in an article titled “Three-Dimensional Particle Tracking Via Bifocal Imaging” by E Toprak et al. (Nano Lett., 2007, 7(7): pp. 2043-45), which is incorporated by reference in its entirety. As such, advances in temporal resolution to sub-millisecond levels have been limited only to 2D imaging.
Thus, there is a need for a microscopy system that can provide 3D imaging with resolution below 100 nanometers in all three dimensions. Another need is directed to achieving particle-tracking in 3D with a temporal resolution below 1 millisecond for enabling visualization of dynamic sub-cellular processes. The present invention is directed to satisfying one or more of these needs and solving other problems.